Hepatitis C virus (HCV) infection persists for years in most patients and leads to chronic liver disease despite robust immune responses. Although there are no clearly established in vitro correlates of protective immunity, multiple lines of evidence suggest that CD4+ and CD8+ T cell responses, while critical for controlling acute infection, are insufficient for prevention of long-term persistence. At the same time, emerging evidence supports the importance of virus neutralizing (Vn) antibodies, and the ability of B cell responses to modify the course of infection. Thus, an effective vaccine will need to induce both robust T cell as well as B cell responses. To design a vaccine immunogen capable of eliciting broadly reactive, Vn antibodies against this highly diverse virus, it is critically important to better define the immunogenic determinants of HCV. Our hypothesis is that these Vn epitopes are organized in discrete clusters (Vn domains) and that specific domains either participate directly in attachment or binding to viral co-receptors, or in the conformational changes required for viral maturation or entry. We also propose that certain overlapping Vn epitopes are likely to be more conserved and less capable of mutations leading to virus escape. This proposal brings together two highly productive and complementary research teams in an effort to test these hypotheses and develop critically needed information on the structure of the Vn epitopes of HCV. Characterization of multiple human monoclonal antibodies (HMAbs) to HCV isolated by the Foung laboratory has revealed the existence of multiple immunogenic domains on E2, at least three of which mediate Vn by blocking E2 binding to CD81, an essential co-receptor for HCV entry. Complementary work in the Lemon laboratory has led to the development of novel cell culture-permissive viruses allowing in vitro characterization of Vn and viral escape from Vn antibodies. Our immediate aims are to exploit these novel tools and to identify broadly conserved, immunogenic domains on the HCV envelope that elicit Vn antibodies. We will determine which envelope protein domains are involved in specific phases of virus entry, and which epitopes within each domain are recognized by broadly Vn antibodies and are less capable of sustaining escape mutations. This will be accomplished through further studies of an extensive panel of Vn HMAbs, generation of new Vn HMAbs, and the in vitro selection and characterization of Vn escape mutants using a cell culture-infectious HCV chimera with structural proteins derived from genotype 1a H77c virus. These novel escape mutants will be utilized as antigen for selection of additional HMAbs. Collectively, these studies will create a high-resolution, functional map of conformational Vn epitopes comprising the major binding sites of both genotype-specific and broadly Vn antibodies on the HCV envelope. The information gained from these efforts will provide the basis for rational vaccine design, and provide much needed insight into the molecular specificities of antibodies that should be elicited by immunization or that would be useful for immunotherapy